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According to the USDA’s National Agricultural Statistics Service (NASS), corn was the largest crop in America in 2019 with 91.7 million acres planted (USDA, National Agricultural Statistics Service, 2019a). Corn was also the most planted crop in South Carolina, with 380,000 acres planted in 2019 (USDA, National Agricultural Statistics Service, 2019b). Corn has many known pests and the most common nematodes affecting this valuable crop are spiral and root-lesion nematodes, followed by dagger, needle, ring, stunt, pin, lance, and stubby root nematodes (Tylka et al., 2011; Yan et al., 2016).
The Seville root-knot nematode, Meloidogyne hispanica (Hirschmann, 1986) was studied for the first time by Dalmasso and Bergé (1978) from Seville, Spain from peach rootstock (Prunus persica silvestris Batsch) and later described as M. hispanica by Hirschmann (1986). The species has been reported infecting many economically important crops such as tomato, beet, corn, pepper, cucumber, eggplant, potato, bean, and others in several countries of Europe, Asia, Africa, Central and South America (Maleita et al., 2012a, 2012b; Subbotin et al., 2021). Until now, there have been no verified reports of this nematode in North America. Meloidogyne hispanica belongs to the Ethiopica group of root-knot nematodes, which also contains the species M. ethiopica, M. luci and M. inornata (Alvarez-Ortega et al., 2019).
The objective of this work was to provide morphological and molecular characterization of this root-knot nematode isolated from corn in South Carolina, identified herein as Meloidogyne hispanica. This report represents the first record of this species in the USA and North America.
Materials and methods
Two soil samples collected from a corn field from Pickens County, South Carolina were sent by Diana Low (Clemson University) to the Mycology and Nematology Genetic Diversity and Biology Laboratory (MNGDBL), Beltsville, MD in fall of 2019 and early 2020. Nematodes were extracted from soil using sugar centrifugal flotation method (Jenkins, 1968). For morphological study nematodes were fixed in 3% formaldehyde and processed to glycerin by the formalin glycerin method (Hooper, 1970; Golden, 1990). Photomicrographs of the specimens were made with a Nikon Eclipse Ni compound microscope using a Nikon DS-Ri2 camera. Measurements were made with an ocular micrometer on a Leica WILD MPS48, Leitz DMRB compound microscope.
For molecular identifications, single nematodes were mechanically disrupted with a micro knife in 20 µl nematode extraction buffer (500 mM KCl, 100 mM Tris-Cl (pH8.3), 15 mM MgCl2, 10 mM dithiothreitol (DTT), 4.5% Tween 20 and 0.1% gelatin) (Thomas et al., 1997) and stored at −80°C until needed. To prepare DNA extract, frozen nematodes were thawed, 1 µl proteinase K (from 2 mg/ml stock solution) was added, and the tubes were incubated at 60°C for 60 min, followed by 95°C for 15 min to deactivate the proteinase K. Two or three microliters of extract were used for each PCR reaction. Six J2 were examined for each marker.
DNA markers were amplified using the following primers: the internal transcribed spacer region (ITS1-5.8S-ITS2) of rRNA gene was amplified with primers TW81 [5′-GTTTCCGTAGGTGAACCTGC-3′] and AB28 [5′-ATATGCTTAAGTTCAGCGGGT-3′] as described by Skantar et al. (2012); the D2–D3 expansion segments of the large subunit (LSU) 28S rRNA gene were amplified with primers D2A [5′- ACAAGTACCGTGAGGGAAAGTT-3′] and D3B [5′-TCGGAAGGAACCAGCTACTA-3′] according to De Ley et al. (2005). The mitochondrial nad5 region was amplified with primers NAD5F2 [5′-TATTTTTTGTTTGAGATATATTAG-3′] and NAD5R1 [5′-TATTTTTTGTTTGAGATATATTAG-3′] as described (Janssen et al., 2016); the mtDNA intergenic COII-16S region was amplified with primers C2F3 [5′-GGTCAATGTTCAGAAATTTGTGG-3′] and 1108 [5′-TACCTTTGACCAATCACGCT-3′] as described by Powers and Harris (1993); and mitochondrial COI was amplified with primers JB3 [5′-TTTTTTGGGCATCCTGAGGTTTAT-3′] and JB5 [5′-AGCACCTAAACTTAAAACATAATGAAAATG-3′] according to Derycke et al. (2010).
All PCR products were cleaned with the Monarch DNA Gel Extraction Kit (NEB, Ipswitch, MA). The ITS rRNA gene fragments were cloned using the Strataclone PCR Cloning Kit (Agilent, Santa Clara, CA); cleaned amplicons were sequenced directly by Genewiz, Inc. Cloned plasmid DNA was prepared with the Monarch Plasmid Miniprep Kit (NEB) and sequenced. Distinct sequences obtained from as many as six J2 were submitted to GenBank as follows: M. hispanica: 28S rRNA gene, MZ328884, MZ328885; COI gene, MZ332972-MZ332973; nad5 gene, MZ332520-MZ332521; COII-16S, region MZ332519; ITS clone sequences came from specimens 112C5 (MZ328463, MZ328455), 112C6 (MZ328459, MZ328461), 112C7 (MZ328456, MZ328457), 112E1 (MZ328452, MZ328460), 112E2 (MZ328458, MZ328454), 112E3 (MZ328453, MZ328462).
Alignments with the ITS rRNA, D2-D3 of 28S rRNA, COI, nad5 gene sequences and intergenic COII-16S gene region of mtDNA of new sequences M hispanica with other sequences of this species and other root-knot nematodes were created using ClustalX 1.83 (Chenna et al., 2003) with default parameters. The alignments for ITS rRNA, D2-D3 of 28S rRNAand COII-16S gene sequences were used to construct phylogenetic networks using statistical parsimony (SP) as implemented in POPART software (http://popart.otago.ac.nz) (Bandelt et al., 1999).
Results and discussion
Meloidogyne hispanica measurements
Second stage juveniles (n = 10) L = 342.0 ± 14.6 (325.0–352.0) μm, stylet = 10.5 ± 0.6 (10.0–11.5) μm, tail length = 41.7 ± 1.2 (40.0–43.0) μm; hyaline region = 12.8 ± 1.5 (10.0 –15.0) μm; a = 26.8 ± 1.4 (25.0–29.0); b = 2.9 ± 0.1 (2.7–3.2); c = 8.3 ± 0.5 (7.8–9.4).
Meloidogyne hispanica descriptions
Second-stage juveniles (Fig, 1). Body annules distinct, becoming irregular in posterior tail region. Four incisures in lateral field, outer lines crenate. Labial region truncate, offset from body. Stylet delicate, with cone sharply pointed, increasing in width posteriorly, shaft cylindrical, widens slightly posteriorly, knobs robust, distinctly separated, rounded, and sloping posteriorly. Pharyngo-intestinal junction indistinct, near nerve ring. Pharyngeal gland lobe variable in length. Hemizonid 2 annuli anterior to excretory pore. Tail slender, with a bluntly rounded terminus. Posterior tail region, with large annules of variable size. Hyaline region indistinct. Rectal dilation large. Phasmids obscure but located a short distance posterior to anal opening. The body mean length measurements for this isolate are shorter from the original description 392.0 (356.4–441.4) μm. The mean values of the stylet length and tail length are also shorter from the population described by Hirschmann (1986) but still within the range. All other morphology and morphometrics of the South Carolina population fit very well with the type specimens from the original description.
Figure 1:
Photomicrographs of Meloidogyne hipsanica juveniles. (A–C) anterior end; (D) lateral field; (E, F) posterior ends with arrow pointing the anal area (E).
From Meloidogyne luci (Carneiro et al., 2014) juveniles are different by having a shorter stylet length (10.0–11.5 vs 12–13.5 μm) and by the shape of the tail and tail terminus which slender, with a bluntly rounded terminus vs conoid tail with finely rounded unstriated terminus.
Molecular characterization
The partial COI gene
The partial COI gene sequence of M. hispanica was identical to those of M. hispanica (JX683712, JX683713) from China (Wang et al., unpublished), M. javanica, M. incognita, M. arenaria, M. floridensis and several other species. It differed in one nucleotide from COI sequence of M. luci.
The partial nad5 gene
The partial nad5 gene sequence was identical to that of M. arenaria (H3) M. inornata and M. ethiopica (Janssen et al., 2016).
The D2-D3 of 28S rRNA gene
The D2–D3 of 28S rRNA gene alignment included 89 sequences and was 457 bp in length. The M. hispanica sequence from South Carolina (MZ328885) formed a separate group with other representatives of M. hispanica, M. luci, M. ethiopica and two sequences identified as M. incognita (Fig. 2).
Figure 2:
Statistical parsimony network showing the phylogenetic relationships between the D2–D3 of 28S rRNA gene sequences of Meloidogyne species from the tropical RKN complex. The sequences of each species are marked by different colors. Pies (circles) represent sequences of each species with the same haplotype and their size is proportional to the number of these sequences in the samples. Numbers of nucleotide differences between the sequences are indicated on lines connecting the pies. Small black dots represent missing haplotypes.
The ITS rRNA gene
The ITS rRNA gene alignment included 50 sequences and was 493 bp in a length. Twelve new sequences of the ITS rRNA gene clones from M. hispanica were distributed among the tropical RKN complex: (i) three sequences within the Incognita group; (ii) seven sequences within the Ethiopica group and two sequences formed a separate group (Fig. 3). Sequence variation between ITS rRNA gene clones from South Carolina M. hispanica reached 11.5%.
Figure 3:
Statistical parsimony network showing the phylogenetic relationships between the ITS rRNA gene sequences of Meloidogyne species from the tropical RKN complex. The sequences of each species are marked by different colors. Pies (circles) represent sequences of each species with the same haplotype and their size is proportional to the number of these sequences in the samples. Numbers of nucleotide differences between the sequences are indicated on lines connecting the pies. Small black dots represent missing haplotypes. * - identified as Meloidogyne incognita, but maybe belong to M. hispanica.
The intergenic COII-16S gene region
The COII-16S gene alignment included 37 sequences and was 1506 bp in length. Analysis of the intergenic region clearly differentiated M. hispanica from other related species (Fig. 4). The sequence of the South Carolina population clustered with sequence of M. hispanica from Portugal (Maleita et al., 2012a, 2012b) and sequence of Meloidogyne sp. from Australia (Fargette et al., 2010) identified here as a representative of this species.
Figure 4:
Statistical parsimony network showing the phylogenetic relationships between intergenic COII-16S gene sequences of Meloidogyne species having a long intergenic region. The sequences of each species are marked by different colors. Pies (circles) represent sequences of each species with the same haplotype and their size is proportional to the number of these sequences in the samples. Numbers of nucleotide differences between the sequences are indicated on lines connecting the pies. Small black dots represent missing haplotypes. * - identified in GenBank as Meloidogyne sp.
Network analysis using statistical parsimony method was applied here for the study of relationships of root-knot nematodes. This method is especially useful for analysis of the datasets containing similar sequences. Moreover, the network can predict haplotypes and reveal sites where recombination and sequence errors are likely to have occurred. Since the network harbors all trees for the input data, it yields a more concise picture of relationships. Thus, our study showed that the ribosomal rRNA (ITS and D2–D3 of 28S) and mitochondrial COI and nad5 gene sequences did not distinguish M. hispanica from other root-knot nematode species. Only the intergenic COII-16S gene region sequence allows clear separation of M. hispanica from closely related species.
To our knowledge the finding of Meloidgyne hispanica in South Carolina represents the first report of this species in the United States and North America. Reproduction of M. hispanica was previously evaluated on 63 cultivated host plants, revealing a broad host range (Maleita et al., 2012a, 2012b). Meloidogyne hispanica was most recently reported on corn in Greece in 2017 (Tzortzakakis et al., 2019) and on sunflower in Greece in 2013 (Tzortzakakis et al., 2014). The threat of damage caused by M. hispanica to corn or other susceptible crops in the U.S. remains to be determined but could become significant if present under drought conditions. Some degree of control may be achievable through rotation with pepper cultivars containing resistance genes (Maleita et al., 2012a, 2012b). Continued monitoring to limit further spread of M. hispanica is needed.
